Poly acrylamide gel electrophoresis (PAGE

Nucleartranslocationofp-Catenin

Somatoplot Coordinates

The method followed is essentially the same as described by Jin et al. (1992). Overnight bacterial cultures grown in minimal A medium supplemented with 0.4% glycerol and 0.5% Casamino acids with the appropriate antibiotic were subcultured 1:100 in the same medium in a volume of 20 ml (0.2% arabinose was added for induction of the plasmid-borne gene downstream of Para, wherever required) at 37 ̊C. Cultures were induced with 1 mM IPTG at A600=0.3. 1 ml samples were aliquoted at time intervals of 0 sec, 20 sec, 40 sec, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min and 6 min into 1 ml of 0.1 mg/ml ice cold chloramphenicol and the samples were put on ice. After sampling the cultures were incubated at 37 ̊C for 15 min. 0.5 ml of this culture was then taken in duplicate tubes for β-galactosidase assays

RNA polymerase elongation rate measurement

Log-phase yeastcells were collected, washed and suspendedin 10 mM Tris-HCl (pH 7.5) containing 50 mg/ml zymolyase-20T. Cell suspension was incubated at room temperature and absorbance was monitored at 600 nm every10mininterval. Initial absorbance of the cultures at 0 minwas normalized to 100%and the graph was plottedas%decrease in the absorbance with respect to time

Zymolyasedigestion assay

Resultant precipitate was dissolved in 3 N HCl and reprecipitated in methanol:acetic acid (8:1) solution. Following 16 h incubation at room temperature, the precipitate was washed withmethanol:acetic acid (8:1) solution till green colour of the supernatant disappeared.Finally,pellet was washed thrice with methanol and air dried. Driedpellet was resuspended in 0.5 NHCl and total mannan content was quantified with phenol-sulphuric acid carbohydrate estimation method as described earlier.Commercially available purified glucose was used as the standard

Total mannan from 3% NaOH-extractable supernatant of cell wall was precipitated by Benedict’s solution.Reducing sugars(mostly mannan) from alkali-extractable supernatant reactwith copper(II) sulphate present in Benedict’s solution and forms red copper(I) oxide precipitate.Briefly, equal volume of Benedict’s solution was added to 3% NaOH-extractable cell wall supernatant fraction and heated at 99 ̊C for 10 min

Total mannan estimation

Cell wall β-glucan measurement was carried out as describedpreviously with some modifications(Kapteynet.al.,2001). Briefly, cell wall fractions were washed multiple times with 1 N NaCl. Washed cell walls were boiled twice in 50 mM Tris-HCl(pH 7.8) containing 2% SDS, 100 mM Na-EDTA and 40 mM β-mercaptoethanol for 5 min to remove non-covalently linked proteins and other contaminants. SDS-treated cell wall fraction was collected and rinsed thrice with water. For β-glucan isolation, cell wallswere extracted three times, each for 1 h, in 0.5 ml 3% NaOH at 75 ̊C and centrifuged at 1,200 g.All 3% NaOH supernatant fractions were saved for isolation of mannan as described below. 3% NaOH-extractable cell wall pelletwasneutralized twice in 100 mM Tris-HCl (pH 7.5) and once in 10 mM Tris-HCl (pH 7.5) and digested with 5 mg/ml zymolyase-20T in 10 mM Tris-HCl (pH 7.5) for 14-16 h at 37 ̊C. This treatment liberates approximately 90-95% glucose into the supernatant. Total glucan content in the cell wall was measured by estimating glucose from both the solubilised supernatant and zymolyase-20T insoluble pellet fractions with phenol-sulphuric acid carbohydrate estimation method using purified glucose as the standard

Total β-glucan estimation

min. Cells were normalized to equal OD600, resuspendedin 1 ml 50 mM Tris-HCl (pH 7.5) and transferred to 2 ml microcentrifuge tubes. Cells were lysed with glass beadsin a homogenizer (FastPrep®-24,MP Biomedicals)asdescribed earlier.Brokencells were washed from glass beadswith 500 μl Tris-HCl (50 mM, pH 7.5) and pelleteddown at 15,000 g for 10 minto obtainall cell wall and membrane content. Pellet was then boiled for 10 minin 1mlTris-HCl(50mM; pH 7.5)solutioncontaining 2%SDS. SDS-extractable material(mannoproteins)was savedand remaining pellet wasboiled again in 500 μl Tris-HCl(50 mM; pH 7.5)buffer containing 2%SDS. Cell wallwas collectedby centrifugation at 15,000 g for 10 min, washed twice with1 ml waterandresuspendedin 100 μl 67 mM potassium phosphatebuffer. This washed cell wall materialwas used for β-glucan estimation as described below

Yeast cell wall was isolatedas describedpreviously(De Groot et al., 2004). Briefly, cells grown underdifferent environmental conditions were harvested at 5,000 g for 5

Crude cell wall isolation

Cell wall isolation, zymolyasedigestion assay and β-glucan estimation

The quantityand purity of nucleic acids weredetermined by measuring the absorbance at 260 and 280 nmusing automated NanoDrop instrument. The concentration of nucleic acids was calculated by taking 1 OD at 260nm = 50μg/ml for DNA and 40 μg/ml for RNA. The purity of nucleic acids was checked by their A260/A280 ratio

Spectrophotometric estimation of nucleic acids

A single colony of E. coli DH5α strain was inoculated in 10 mL LB medium and incubated at 37°C overnight. Overnight culture was subculturedin 250 mL of LB medium of about 0.1 OD and incubated at 18°Cfor36 htillthe OD600reached approximately0.5. Cultures were centrifuged at 2,500 x gfor 10 min at 4 ̊C and harvested cells were washed gently with 80 ml ice-cold Inoue transformation buffer(Section 2.1.6.2). Cells were collected by centrifugation at 2,500x g for 10 min at 4 ̊C and gently resuspended in 20 mLice-cold Inoue transformation buffer. To this cell suspension, 1.5 ml sterile DMSO was added and swirled gently. The cell suspension waskept on ice for 30min,and 100 μLvolume was aliquoted into pre-chilled sterile microcentrifuge tubes. Cells were immediately snap-frozen in liquid nitrogen and stored at -80°C

Preparation of E. coli DH5α ultracompetent cells

Ip6k1-/-(4 male, 5 female)] were exposed to 4NQO (100 μg/mL) in their drinking water. Mice were allowed free access to drinking water containing the carcinogen, and the water was changed every week.Water bottles containing carcinogen was covered with foil to avoid exposure to light. All the apparatus used for this study and remaining water were decontaminated using 1% sodiumhypochlorite for 30 min. Every week water consumption and weight of the each animal was monitored. Mice displaying any characteristics of weight loss or dehydration before 24 weeks were euthanized and examined for lesions. After 24 weeks, mice were euthanized by CO2inhalation and a complete necropsy was performed. Tissues from the aerodigestive tract (tongue, esophagus, stomach,and duodenum) werefixed in formalin and paraffin-embedded sections were stained with hematoxylin and eosin (H&E) to examine thelesions by light microscopy.Images were acquired using a bright field light microscope (Nikon ECLIPSE Ni, NIS Elements acquisition software, 20x 0.5 N.A. objective). During the pathologicalexamination, the lesions observed in the various tissues were categorized into hyperplasia, dysplasia,and invasive carcinoma. Hyperplasia was defined as an increase in the layers or thickening of the epithelium with hyperkeratinization. Dysplasia was defined as loss of epithelial cell polarity, nuclear pleomorphism and abnormal mitoses confined to the epithelium. Invasive carcinoma was defined as invasion of dysplastic cells or lesions into subepithelial tissues such as submucosa and muscle

Carcinogenesis studies were conducted as described previously for Ip6k2-/-mice (Morrisonet al., 2009). Briefly, the carcinogen, 4NQO stock solution (5 mg/mL) was prepared fresh in propylene glycol every week. Four-week old mice [11 Ip6k1+/+(5 male, 6 female) and 9

Carcinogenesis studies